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Targeting T Cells for the Immune-Modulation of Human Diseases by Regina Lin Department of Immunology Duke University Date:___

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Targeting T Cells for the Immune-Modulation of Human Diseases by Regina Lin Department of Immunology Duke University Date:___ Targeting T Cells for the Immune-Modulation of Human Diseases by Regina Lin Department of Immunology Duke University Date:_______________________ Approved: ___________________________ Qi-Jing Li, Supervisor ___________________________ You-Wen He, Chair ___________________________ Michael S. Krangel ___________________________ Xiaoping Zhong ___________________________ Xiao-Fan Wang Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Immunology in the Graduate School of Duke University 2015 ABSTRACT Targeting T Cells for the Immune-Modulation of Human Diseases by Regina Lin Department of Immunology Duke University Date:_______________________ Approved: ___________________________ Qi-Jing Li, Supervisor ___________________________ You-Wen He, Chair ___________________________ Michael S. Krangel ___________________________ Xiaoping Zhong ___________________________ Xiao-Fan Wang An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Immunology in the Graduate School of Duke University 2015 Copyright by Regina Lin 2015 Abstract Dysregulated inflammation underlies the pathogenesis of a myriad of human diseases ranging from cancer to autoimmunity. As coordinators, executers and sentinels of host immunity, T cells represent a compelling target population for immune- modulation. In fact, the antigen-specificity, cytotoxicity and promise of long-lived immune-protection make T cells ideal vehicles for cancer immunotherapy. Interventions for autoimmune disorders, on the other hand, aim to dampen T cell-mediated inflammation and promote their regulatory functions. Although significant strides have been made in targeting T cells for immune-modulation, current approaches remain less than ideal and leave room for improvement. In this dissertation, I seek to improve on current T cell-targeted immunotherapies, by identifying and preclinically characterizing their mechanisms of action and in vivo therapeutic efficacy. CD8+ cytotoxic T cells have potent antitumor activity and therefore are leading candidates for use in cancer immunotherapy. The application of CD8+ T cells for clinical use has been limited by the susceptibility of ex vivo-expanded CD8+ T cells to become dysfunctional in response to immunosuppressive microenvironments. To enhance the efficacy of adoptive cell transfer therapy (ACT), we established a novel microRNA- targeting approach that augments CTL cytotoxicity and preserves immunocompetence. Specifically, we screened for miRNAs that modulate cytotoxicity and identified miR-23a as a strong functional repressor of the transcription factor Blimp-1, which promotes CTL cytotoxicity and effector cell differentiation. In a cohort of advanced lung cancer patients, iv miR-23a was upregulated in tumor-infiltrating CD8+ T cells, and its expression correlated with impaired antitumor potential of patient CD8+ T cells. We determined that tumor-derived TGF-β directly suppresses CD8+ T cell immune function by elevating miR-23a and downregulating Blimp-1. Functional blockade of miR-23a in human CD8+ T cells enhanced granzyme B expression; and in mice with established tumors, immunotherapy with just a small number of tumor-specific CD8+ T cells in which miR- 23a was inhibited robustly hindered tumor progression. Together, our findings provide a miRNA-based strategy that subverts the immunosuppression of CD8+ T cells that is often observed during adoptive cell transfer tumor immunotherapy and identify a TGFβ- mediated tumor immune-evasion pathway. Having established that miR-23a-inhibition can enhance the quality and functional-resilience of anti-tumor CD8+ T cells, especially within the immune- suppressive tumor microenvironment, we went on to interrogate the translational applicability of this strategy in the context of chimeric antigen receptor (CAR)-modified CD8+ T cells. Although CAR T cells hold immense promise for ACT, CAR T cells are not completely curative due to their in vivo functional suppression by immune barriers ‒ such as TGFβ ‒ within the tumor microenvironment. Since TGFβ poses a substantial immune barrier in the tumor microenvironment, we sought to investigate whether inhibiting miR-23a in CAR T cells can confer immune-competence to afford enhanced tumor clearance. To this end, we retrovirally transduced wildtype and miR-23a-deficient CD8+ T cells with the EGFRvIII-CAR, which targets the PepvIII tumor-specific epitope v expressed by glioblastomas (GBM). Our in vitro studies demonstrated that while wildtype EGFRvIII-CAR T cells were vulnerable to functional suppression by TGFβ, miR-23a abrogation rendered EGFRvIII-CAR T cells immune-resistant to TGFβ. Rigorous preclinical studies are currently underway to evaluate the efficacy of miR-23a- deficient EGFRvIII-CAR T cells for GBM immunotherapy. Lastly, we explored novel immune-suppressive therapies by the biological characterization of pharmacological agents that could target T cells. Although immune- suppressive drugs are classical therapies for a wide range of autoimmune diseases, they are accompanied by severe adverse effects. This motivated our search for novel immune- suppressive agents that are efficacious and lack undesirable side effects. To this end, we explored the potential utility of subglutinol A, a natural product isolated from the endophytic fungus Fusarium subglutinans. We showed that subglutinol A exerts multimodal immune-suppressive effects on activated T cells in vitro: subglutinol A effectively blocked T cell proliferation and survival, while profoundly inhibiting pro- inflammatory IFNγ and IL-17 production by fully-differentiated effector Th1 and Th17 cells. Our data further revealed that subglutinol A might exert its anti-inflammatory effects by exacerbating mitochondrial damage in T cells, but not in innate immune cells or fibroblasts. Additionally, we demonstrated that subglutinol A significantly reduced lymphocytic infiltration into the footpad and ameliorated footpad swelling in the mouse model of Th1-driven delayed-type hypersensitivity. These results suggest the potential of subglutinol A as a novel therapeutic for inflammatory diseases. vi Dedication To Mom, Dad, Jose and Bacon. vii Contents Abstract .............................................................................................................................. iv List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Acknowledgements ......................................................................................................... xvii 1. Introduction ..................................................................................................................... 1 1.1 Cancer immunotherapy: An overview .................................................................... 2 1.1.1 Hurdles to successful cancer immunotherapy .................................................... 4 1.1.2 Cytokine-based approaches.............................................................................. 10 1.1.3 Antibody-based approaches ............................................................................. 15 1.1.4 Cell-based approaches...................................................................................... 18 1.2 CD8+ T cells in cancer immunotherapy ............................................................... 23 1.2.1 Immune-protection conferred by effector and memory CD8+ T cells ............ 23 1.2.2 Anti-tumor effector functions of CD8+ T cells ............................................... 26 1.2.3 Transcriptional regulation of CD8+ T cell effector functions and memory generation .................................................................................................................. 31 1.3 Chimeric antigen receptors (CARs): Novel gene therapy tools for ACT ............. 36 1.3.1 Utility of CAR T cell-based ACT .................................................................... 37 1.3.2 Current challenges facing CAR T cell-based ACT .......................................... 39 1.4 MicroRNAs as targets for T cell immune-modulation ......................................... 41 1.4.1 Roles of microRNAs in CD8+ T cell fate and function ................................... 41 1.4.2 Tools for targeting microRNAs ....................................................................... 43 viii 1.5 Targeting auto-reactive effector T cells for the treatment of inflammatory diseases ..................................................................................................................................... 46 1.5.1 The unmet need for tolerable T cell-specific immune-suppressants ............... 46 2. Materials and Methods .................................................................................................. 49 2.1 Mice ...................................................................................................................... 49 2.2 Cell culture ............................................................................................................ 49 2.3 miRNA expression profiling and miRNA qPCR .................................................. 50 2.4 Target prediction and luciferase reporter assays ................................................... 51 2.5
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